The long-term goal of our research is to understand the genetics and evolution of reproductive isolation in natural populations of sexually-reproducing organisms: i.e., the origin of species. This proposal focuses on the genetics of prezygotic reproductive isolation in wild populations of two sister species of monkeyflower, Mimulus lewisii, and M. cardinalis. Previous studies have demonstrated that reproductive isolation between sympatric populations of these species is mediated almost exclusively by the fidelity of their different pollinator guilds. M. lewisii is pollinated by bumblebees, M. cardinalis is pollinated by hummingbirds, and pollinator preference depends strongly on flower color differences between them. The Specific Aims of this project are to: Aim 1. Produce high-resolution genetic and physical maps of the 4 major quantitative trait loci (QTL) controlling differences in flower color between bumblebee-pollinated M. lewisii and hummingbird-pollinated M. cardinalis. Physical regions containing these QTLs will be sequenced an annotated. Aim 2. Construct and characterize high-resolution near-isogenic lines (hrNILs) for each of the 4 major loci controlling differences in flower color between M. lewisii and M. cardinalis. hrNILs will approximate single-gene substitutions, and will be suitable for field experiments. Aim 3. Determine the proportion of the total prezygotic reproductive isolation between M. lewisii and M. cardinalis attributable to each of the 4 flower color loci, and to their combinatorial effects, by direct observation of hrNILs in common garden field experiments in areas of natural sympatry. The effect of each of the 4 flower color loci (in all 15 possible combinations) on pollinator preference will be assessed in field experiments with arrays of hrNILs and the ancestral M. lewisii. A final experiment will directly compare the preference of bumblebees and hummingbirds for the ancestral M. lewisii phenotype, a 4-locus hrNIL in a M. lewisii background, and the derived M. cardinalis. The pollinator visitation phenotypes of all 15 hrNIL combinations, combined with data from parental M. lewisii and M. cardinalis, will allow us to estimate the individual and aggregate effect of all 4 loci on reproductive isolation. Our expectation is that the vast majority (>75%) of reproductive isolation in sympatry will be attributable to just these 4 flower color loci. These experiments will provide our first glimpse into the multi-locus genetics of prezygotic reproductive isolation in any natural system. PUBLIC HEALTH RELEVANCE: The long-term goal of our research is to understand the genetics and evolution of reproductive isolation in natural populations of sexually reproducing organisms: i.e., the origin of species. All of the biodiversity on Earth, including the origin of humans, is a result of the process of speciation.